Biological macromolecular functions require distinct conformational states that are challenging to examine comprehensively. Current methods to quantify conformational similarities and distinguish different assembly states are underdeveloped. Recent developments in small-angle X-ray scattering (SAXS) have shown that SAXS can provide the resolution to resolve conformational states, characterize flexible macromolecules and screen in high throughput under most solution conditions1. However, robust tools for comprehensively characterizing and visualizing the different conformational states identified by SAXS have been lacking. Here we present the SAXS structural comparison map (SCM) and volatility of ratio (V_R) difference metric, which together provide quantitative and superposition-independent evaluation of solution-state conformations.

read more in the full article…

Small-angle X-ray scattering (SAXS) is a robust technique for the comprehensive structural characterizations of biological macromolecular complexes in solution. Here, we present a coherent synthesis of SAXS theory and experiment with a focus on analytical tools for accurate, objective, and high-throughput investigations. Perceived SAXS limitations are considered in light of its origins, and we present current methods that extend SAXS data analysis to the super-resolution regime. In particular, we discuss hybrid structural methods, illustrating the role of SAXS in structure refinement with NMR and ensemble refinement with single-molecule FRET. High-throughput genomics and proteomics are far outpacing macromolecular structure determinations, creating information gaps between the plethora of newly identified genes, known structures, and the structure-function relationship in the underlying biological networks. SAXS can bridge these information gaps by providing a reliable, high-throughput structural characterization of macromolecular complexes under physiological conditions.

“In SAS imaging, beams of X-rays or neutrons sent through a sample produce tiny collisions between the X-rays or neutrons and nano- or subnano-sized particles within the sample. How these collisions scatter are unique for each particle and can be measured to determine the particle’s shape and size. The analytic metrics developed by Rambo and Tainer are predicated on the discovery by Rambo of an SAS invariant, meaning its value does not change no matter how or where the measurement was performed. This invariant has been dubbed the “volume-of-correlation” and its value is derived from the scattered intensities of X-rays or neutrons that are specific to the structural states of particles, yet are independent of their concentrations and compositions.”

MW, molecular mass. V_c and R_g were determined from theoretical atomic X-ray scattering profiles for 9,446 protein PDB structures. For each profile, SAXS data were simulated to a maximum q = 0.5 Å⁻¹ (~13 Å). Various ratios of V_c and R_g against protein mass were examined in a log-log plot. The linear relationship observed for the ratio V_c²R_g⁻¹ (black) suggests that a power-law relationship exists between the ratio and particle mass of the form ratio = c(mass)^k. The ratio, V_c²R_g⁻¹, is defined by units of ų with mass in Daltons. Additional ratios examined (green, cyan, grey and red) displayed asymmetric nonlinear relationships.

The SIBYLS beamline (12.3.1) of the Advanced Light Source at Lawrence Berkeley National Laboratory, supported by the US Department of Energy and the National Institutes of Health, is optimized for both small-angle X-ray scattering (SAXS) and macromolecular crystallography (MX), making it unique among the world’s mostly SAXS or MX dedicated beamlines. Since SIBYLS was commissioned, assessments of the limitations and advantages of a combined SAXS and MX beamline have suggested new strategies for integration and optimal data collection methods and have led to additional hardware and software enhancements. Features described include a dual mode monochromator [containing both Si(111) crystals and Mo/B₄C multilayer elements], rapid beamline optics conversion between SAXS and MX modes, active beam stabilization, sample-loading robotics, and mail-in and remote data collection. These features allow users to gain valuable insights from both dynamic solution scattering and high-resolution atomic diffraction experiments performed at a single synchrotron beamline. Key practical issues considered for data collection and analysis include radiation damage, structural ensembles, alternative conformers and flexibility. SIBYLS develops and applies efficient combined MX and SAXS methods that deliver high-impact results by providing robust cost-effective routes to connect structures to biology and by performing experiments that aid beamline designs for next generation light sources.

Classen, S, Hura, GL, Holton, JM, Rambo, RP, Rodic, I, McGuire, PJ, Dyer, K, Hammel, M, Meigs, G, Frankel, KA, and Tainer, JA “Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source.” (2013). J Appl Crystallogr 46, 1-13.

The point-spread function (PSF) of a fiber-optic taper-coupled CCD area detector was measured over five decades of intensity using a 20 µm X-ray beam and 2000-fold averaging. The “tails” of the PSF clearly revealed that it is neither Gaussian nor Lorentzian, but instead resembles the solid angle subtended by a pixel at a point source of light held a small distance (27 µm) above the pixel plane. This converges to an inverse cube law far from the beam impact point. Further analysis revealed that the tails are dominated by the fiber-optic taper, with negligible contribution from the phosphor, suggesting that the PSF of all fiber-coupled CCD-type detectors is best described as a Moffat function.

the authors go on to suggest that:

…we expect that by fitting an expression for the spot-PSF convolution as described here directly to pixel values will result in more accurate spot intensity integrals than those currently being obtained using conventional profile-fitting methods (which assume that the intensity of a pixel is due exclusively to X-ray photons falling directly upon it). A “fitting approach” would eliminate systematic errors in background estimation arising from the tails and also suppress the influence of shot noise from X-ray photons falling on pixels outside the “true” spot area.

DOMO is fairly robust, and is capable of handling your precious crystals mounted in a variety of bases:

However, you must take some care when gluing or epoxying the pins into the bases. If there is too much glue or epoxy or you inadvertantly get some on the sides or bottom of the base this will cause the robot to jam, which will require time-wasting reset procedures, lost samples, and unhappy beamline support personnel.

Here is a recent example of several pins where the user (who will remain unnamed) applied entirely too much epoxy. Somehow the user was able to load these pins into the cassette, but they caused the robot to jam.

There are more detailed tips and hints on the SSRL SMB website for preparing your bases and pins.

1. RAPIDD - a rapid access process, replaces the 2-month proposal system. SAXS proposals should use the RAPIDD system. MX applicants may apply for either RAPIDD or 6 Month Proposals.

   The aim is to provide quick turnaround. Proposals are fairly simple, requiring a one page pdf describing the science, and will be accepted at any time. Proposals are sent out for review within two business days, and we hope to complete the review within 2-3 weeks. Beamtime may be allocated at any time after submission depending on your proposal score, the number of proposals submitted, and the beamtime available. We have never rejected a RAPIDD application for SAXS data collection except for applications proposing technically impossible experiments nto suited to the SIBYLS beamline.

2. 6 MONTH PROPOSAL

   This mechanism will suit regular long-term users of the ALS. It has been available since January 2012 and 14 research groups successfully established a 2-year research program in the first cycle. The mechanism allows users to apply for a longer term program through the regular ALS proposal cycle. Proposals are accepted every 6 months, for beamtime starting 4 months later. These proposals may be renewed for subsequent 6 month cycles for up to 2 years. Proposals may cover a broad program of work, and will be submitted as a PDF file, up to 3 pages long. We hope this will reduce the overall workload for users who currently submit more than one proposal a year.

Date: October 9-10, 2012 Location: Advance Light Source (ALS) at Lawrence Berkeley National Laboratory , Berkeley, CA

The SIBYLS team will host a workshop with strong emphasis on experimental aspects of Small Angle X-ray Scattering techniques in structural biology. The two-day workshop will provide training on experimental techniques and software tutorial sessions primarily for biological SAXS studies. One day of the workshop will be dedicated for data processing by workshop participants. The latest advances in SAXS studies on biological systems will be reported and discussed by several experts in a diverse spectrum of structural biology benefiting from bioSAXS (see program bellow). Also planned are presentations on complementary experimental approaches (MODELLER) and solution structure modeling techniques using ensemble analysis. Participants will receive updates on current developments at SIBYLS and development of software dedicated to analyze SAXS for structural biology.

Enrollment is limited to 30 participants. 



Organizers: Michal Hammel, Greg Hura, Robert Rambo



Inquires: Jane Tanamachi

Registration: To attend "Course on SAXS" you need register for the 2012 Advanced Light Source Users' Meeting. ALS user meeting will be held at Berkeley Lab beginning Monday, October 8. "Practical Course on SAXS" will begin Thuesday October 9th and continue through Wednesday October 10th. When you registering, you must indicate "SIBYLS bioSAXS"

Tuesday, October 9th LBNL ( B50 Auditorium )

11:30 Lunch at the ALS patio

12:40 Welcoming Remarks Michal Hammel

12:45 Robert Rambo, LBNL, Berkeley "Small-Angle Scattering and its Application to Soft matter Science: Historical Remarks" "New ways to analyze SAXS from biological material"

13:25 Greg Hura, LBNL, Berkeley "SIBYLS SAXS capabilities and future developments

15:10 Coffee Break

15:20 Alex Grishaev, NIH Title: TBD

16:00 Patrick Weinkam , UCSF "Conformational Sampling to predict SAXS profile: How to use MODELLER"

16:30 Gareth Williams, LBNL "SAXS combined with crystallography and computation: Application of the Ensemble Analysis "

17:00 Michal Hammel , LBNL "Resolution in SAXS"

Wednesday, October 10th LBNL ( B50 Auditorium ),

9:00 Rob Rambo, LBNL, Berkeley "Scatter: New software for SAXS data processing"

9:50 Michal Hammel, LBNL, Berkeley "FOXS-MES-MODELLER: New software for solution structure modeling"

10:30 Coffee Break

10:45 Greg Hura, Robert Rambo and Michal Hammel, LBNL, Berkeley "Data reduction and processing tutorial"

12:00 Lunch Break at the ALS patio

Practical session with Mentors (Greg Hura, Rob Rambo and Michal Hammel)

13:00-17:00 Participating Students "Practical Session - Data reduction and processing"

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.

Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible ”pocket” domain loop such that it mimics and directly blocks E2F transactivation domain (E2FTD) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2FTD-pocket association and protein binding to the pocket ”LxCxE” site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique ‘tyrosine clasp’ enabling endoglycosidic cleavage of branched PAR chains.

Xu, Q. et al. “Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization.” Mol. Microbiol. 83, 712-727 (2012).

The recently identified plant photoreceptor UVR8 triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light via an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine retunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)-a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations